Several characteristics make the liver an attractive target organ for the application of gene therapy. First, the liver is the situs of essential metabolic pathways, and consequently it is often the organ involved in genetic metabolic diseases. Data from model animals suggested that expression of a functional version of a gene in the liver of the affected animal can substantially restore metabolic function. Recent results obtained in human clinical trials have supported this view. The liver is also intimately associated with the circulatory system, and can serve as a secretory organ for the systemic delivery of therapeutic proteins or polypeptides. Another advantage the liver presents as an organ for gene therapy is the particular structure of its endothelium, which permits access of large molecules to the liver parenchyma. Vectors commonly used in gene therapy therefore can penetrate the organ and reach the liver cells.
Liver gene therapy has broad applicability, and can be used in the treatment and prevention of conditions by expression of RNAs, polypeptides or proteins, or combinations thereof, in the liver for action in the liver or secretion of polypeptides or proteins into the bloodstream or the gastrointestinal tract. Conditions that may be treated or prevented by liver gene therapy include, for example, inborn errors of metabolism, lymph and blood protein and polypeptide deficiencies, including for example, clotting abnormalities, obesity, hypercholesterolemia, cystic fibrosis, alpha 1-antitrypsin deficiency, phenylketonuria, diabetes, Wilson disease, ornithine transcarbamylase deficiency, among others, cancer, several forms of hepatitis and other infectious diseases affecting liver, hypertension, cirrhosis of the liver, among others.
Preventive and therapeutic applications of gene transfer to the liver have been recently reviewed in the literature. See, e.g., Davern, et al., Dig. Dis. 16:23-37 (1998); Forbes and Hodgson, Aliment. Pharmacol. Ther. 11:823-836 (1997); Chang and Wu, Gastroenterology 106:1076-1084 (1994); and Horwich, Curr. Top. Microbiol. Immunol. 168:185-200 (1991).
Some liver-directed gene therapy applications require long-term moderate to high level expression of a product encoded by a gene construct. To that end, it has been found desirable that the vector transduce a large proportion of target cells, and that the gene construct stably integrate into the genome of the transduced target cells. Transduction of gene constructs into the genome of liver cells has been accomplished by several means, including the use of retroviral vectors, adeno-associated virus vectors, lipofectant agents, receptor-based transfer, and combinations thereof. See, e.g., Sandig and Strauss, J. Mol. Med. 74:205-212 (1996). Cell division, in particular passage of a cell through the S phase of the cell cycle, has been found to greatly increase, and in some cases has been found indispensable, for integration of a transduced gene construct into a cell's genome or for its adequate expression. See, e.g., Roe, et al., EMBO J. 12:2099-2108 (1993)(retroviruses) and Fisher, et al., Nat. Med. 3:306-312 (1997)(adeno-associated virus). The induction of liver cells to proliferate has also been found to present other advantages for liver-directed gene transfer, including the up-regulation of viral receptor expression by dividing liver cells, which has a positive effect on the efficiency of retroviral vector gene transfer into liver cells. Ott, et al., J. Biol. Chem. 273(19):11954-61 (1998).
Retroviral vectors provide several advantages for in vivo gene transfer, including their ability to carry relatively large amounts of genetic material (8 kilobases (kb)) and their reliable, efficient and consistent integration into a target cell's genome. The use of recombinant retroviruses as gene transfer vectors into liver cells has nevertheless been limited due to methodological difficulties. The well documented quiescent state of liver cells under normal conditions has prevented efficient in vivo liver cell transduction by retroviral vectors because retroviral vectors require division of target cells within a few hours of vector entry for stable integration of the gene construct into the genome of the target cell.
While techniques have been described for inducing the proliferation and transduction of liver cells in vivo in model animals, these techniques have been found inadequate for general use. One presently used strategy, for example, depends on liver regeneration induced by removal of a large portion of the liver (Ferry, et al., Proc. Nat'l Acad. Sci. USA 88:8377-8381 (1991)), by causing apoptosis (cell death) of a large portion of the liver by portal branch occlusion (Bowling, et al., Human Gene Therapy 7:2113-2121 (1996)), or by producing severe liver damage by expression of toxic genes in liver cells (Lieber, et al., Human Gene Therapy 6:1029-37 (1995)). These techniques are laborious, intrusive and may result in complications due to the rapid elimination of a large portion of the liver of an individual. The need for major surgery before integrative transfer is clinically undesirable. Moreover, these techniques are particularly unsuitable for individuals in need of gene therapy due to an already present liver insufficiency.
Less invasive procedures for inducing replication of liver cells have been described, but these procedures have failed to solve the existing problem of low transduction, as they have been found to induce a low number of liver cells to proliferate, and generate an inadequately low proportion of transduced cells. For example, Bosch, et al., (J. Clin. Invest. 98:2683-2687 (1996)) reported that administration of keratinocyte growth factor will induce the proliferation of liver cells. However, only 2% of the hepatocytes in the liver were transduced when a retroviral vector was administered 24 hours after KGF administration.
Kosai, et al., (Human Gene Therapy 9:1293-1301 (1998)) reported that hepatocyte growth factor administration followed by retroviral vector administration will increase transduction of liver cells in vivo. However, low transduction values of 1.3% were achieved. Similarly, Forbes, et al., (Gene Therapy 5:552-555 (1998)) reported that tri-iodothyronine administration induced proliferation of liver cells in vivo, and when followed by retroviral administration increased transduction of liver cells in vivo. Again, however, low transduction values of about 1.3% were achieved.
Therefore, it is desired to provide less invasive and more efficient, effective and convenient compositions and methods for inducing a semi-synchronous wave of liver cell proliferation and for the transduction of an adequate quantity of liver cells without the need for hepatectomy of a large portion of the liver, or otherwise eliminating a large number of liver cells by either expressing a toxic gene in the liver or by causing the apoptotic death of a large number of liver cells by partial portal occlusion.